Multiple lines of evidence suggest that human adipose-derived stromal cells (hASCs) hold promise for future use in craniofacial skeletal regeneration. Human ASCs are easily harvested by simple liposuction procedures and are readily expandable as compared to bone marrow mesenchymal cells. Moreover, human ASCs undergo rapid osteogenic differentiation. In our laboratory, we have observed convincingly that ASCs, whether derived from mouse or human origin, contribute to osseous healing of mouse calvarial defects. For example, a surgical defect in the mouse parietal bone (4mm in diameter) shows no healing when untreated even up to 16 weeks post injury. Upon direct hASC application with an osteoconductive scaffold, significant bony healing is observed in as little as 4 weeks post injury. Perhaps most exciting are those applications of hASCs to the human patient. In small pilot studies, defects of the cranium, maxilla, and mandible have been either healed or enabled to heal faster with the use of hASCs. The current understanding of ASC identity and function, however, remains insufficient to allow larger clinical trials and applications as they are a highly heterogeneous population of cells. While surface antigen expression is an important component to this understanding, it is likely that more in depth analysis will be required to fully characterize hASCs. We aim to provide this analysis through completion of the Specific Aims described in this proposal. We expect the results of these studies to move the field forward by providing a new understanding of the transcriptional diversity within hASCs. We anticipate that the findings from our innovative approach will help to define the relationship between transcriptional activity and cell surface markers on a single cell level. This understanding will allow us to more specifically enrich for a population of cells ideal for craniofacial skeletal regeneration.